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Objective To investigate the impact of radiotherapy simulant Zeocin induced doublestranded RNA (dsRNA)on double-stranded RNA-dependent protein kinase (PKR) activation and explore its molecular mechanism in inhibiting the growth and migration of malignant gliomas. Methods We employed scratch assays, colony formation assays, and CCK8 assays to assess the impact of Zeocin on glioma growth and migration inhibition. Western blot and RT-qPCR were employed to measure the expression levels of methyl-modifying enzymes in glioma cells. The J2 antibody was used to detect endogenous dsRNA in malignant glioma cells treated with Zeocin. Western blot was used to assess the phosphorylation levels of PKR and eukaryotic initiating factor 2α (eIF2α). Results Zeocin significantly inhibited growth and migration of glioma cells; Zeocin treatment induced the production of endogenous dsRNA in malignant glioma cells; as the concentration of Zeocin increased, phosphorylated PKR and eIF2α protein level also significantly increased; Zeocin downregulated the total m6A level of glioma cells in a dosedependent manner; Zeocin selectively downregulated the protein level of methylase METTL14; Zeocin had no effect on mRNA levels of m6A modifying enzymes. Conclusion Zeocin probably triggered more dsRNA by downregulating the m6A level of RNA in malignant glioma cells, which in turn activated the PKR/eIF2α pathway, ultimately leading to tumor growth inhibition. [ABSTRACT FROM AUTHOR]

[Objectives]This study was to explore the molecular mechanisms of increasing of cytokoine induced by Newcastle disease virus (NDV)infection. [Methods]RT-PCR analysis was used to detect mRNA levels of IFN-β,IL-6,IL-8. Western blot analysis and immunofluoresence were used to detect the influence of protein kinases (PKR)and NF-κB pathway during infection. [Results]It was found that IFN-β,IL-6,IL-8 mRNA were significantly up-regulated compared to the uninfected group. And NF-κB pathway was activated from 12 to 24 hours post-infection,with nuclear translocation of p65. Pharmacological inhibition of NF-κB signaling with IKK16 prevented nuclear translocation of p65 and resulted in inhibition of IFN and inflammatory factor expression,demonstrating that NF-κB signaling contributes to this phenomenon. Furthermore,pathogenic pattern recognition receptor PKR was up-regulated and phosphorylated from 12 to 24 hours post-infection. Pharmacological inhibition of PKR kinase activity with 2-aminopurine prevented the nuclear translocation of p65 and inhibited the expression of IFN and inflammatory factor. [Conclusions]NDV induced the increasing of cytokine through activation of PKR-NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Objective: To investigate the way that activated PKR induce the upregulation of P53 function in pancreatic β-cell and the molecular mechanism of the inhibition of the proliferation of pancreatic β-cell. Methods: Pancreatic β-cell was transfected with wt-PKR plasmid and treated with BEPP. Western blot and co-immunoprecipitation was performed to detect P53 protein levels and P53-SUMO-1 protein-protein interaction. Sumoylation inhibitor Spectomycin B1 was pretreated to establish negative control group. Results: In PKR overexpressing β-cells, BEPP induced increase of PKR protein expression but not mRNA expression, and PKR-SUMO-1 protein-protein interaction. Spectomycin B1 inhibited PKR-induced upregulation of P53 sumoylation and protein level. Conclusion: PKR can upregulate the protein level and stability of P53 through P53 sumoylation, involving in pancreatic β-cell dysfunction and themechanism of type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Objective: To investigate the molecular mechanism of apoptosis of HL60 cells induced by oncolytic virus Reovirus type 3 (Reo3)., Methods: HL60 cells were infected with Reo3 at different multiplicity of infection (MOI) with the uninfected HL60 cells as control group. After 48 h of infection, the activity of HL60 cells infected with virus at different MOI was detected by CCK8 method to investigate the influence of MOI to cell activity. Simultaneously, the apoptotic rate of HL60 cells was detected by flow cytometry, and the activation level of double-stranded RNA-dependent protein kinase (PKR) and the expression of apoptotic-related protein in HL60 cells were detected by Western blot. Before infection with Reo3 for 48 h, HL60 cells were treated with 2-aminopurine (2-AP), a specific inhibitor of PKR, for 24 h. Afterward, the apoptotic level and expression of apoptotic related proteins were detected., Results: Activity of HL60 cells was obviously inhibited after infected with Reo3 with a MOI of 1 for 48 h. The cell survival rate was (24.333±3.396)% and the apoptotic rate was (29.96±2.06)%. Both rates were all higher than those in the control group ( P < 0.05). Western blot results showed that the expression levels of PKR, p-PKR, Bax, Caspase3 and cleaved Caspase3 in HL60 cells infected with Reo3 were higher than those in the control group ( P < 0.05), while the expression level of Bcl-2 was lower ( P < 0.05). Compared with the group without inhibitor, the apoptotic rate of HL60 cells pretreated with 2-AP decreased ( P < 0.05), the phosphorylation level of PKR and the expression level of apoptotic-related protein also decreased ( P < 0.05)., Conclusion: Oncolytic virus Reo3 could activate PKR in HL60 cells and thus induce apoptosis of HL60 cells., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)